A primer set, kit and application thereof for high-throughput sequencing targeting pathogens of reproductive tract infections

Abstract

This invention discloses a primer set, kit, and application for high-throughput sequencing of pathogens of reproductive tract infections. This method utilizes multiplex PCR combined with high-throughput sequencing to identify microorganisms and associated drug-resistance genes within the detection range with high sensitivity. The application of this detection technology to the identification of pathogens of reproductive tract infections has important clinical significance, simplifying the operation steps, reducing testing costs, and improving detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a primer set and a kit for high-throughput sequencing of pathogens targeting reproductive tract infections, and applications thereof. Background Art

[0002] Female lower genital tract infections are common gynecological conditions, with vaginitis and cervicitis being the most common. These include, but are not limited to, bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, trichomoniasis, and cervicitis caused by Neisseria gonorrhoeae, Chlamydia, and Mycoplasma. Lower genital tract infections are closely associated with a variety of gynecological and obstetric conditions and can lead to infertility and female genital tract cancers.

[0003] Microorganisms associated with female lower reproductive tract infections include: bacteria, fungi, viruses and special pathogens. The common detection methods currently used in clinical practice are: culture method, morphological detection method, immunological detection method represented by colloidal gold / enzyme-linked immunosorbent assay and common nucleic acid detection method. Among them, the culture method mainly focuses on bacteria and has weak detection capabilities for other types of pathogens, because a large number of pathogens are not easy to survive in an in vitro artificial culture environment; the morphological detection method is mainly based on the typical biological morphology of common pathogens for identification. Although the detection is rapid, the accuracy and stability are poor; the immunological detection method is convenient and fast, with low dependence on professional instruments and personnel, but the accuracy is low and the false negative problem is prominent. In contrast, nucleic acid detection has a better balance between cost, detection speed and detection performance, and has become a mainstream detection method. However, traditional nucleic acid detection technology has gradually failed to meet the clinical detection needs for complex infections, exposing serious shortcomings, including but not limited to: the types and numbers of detected pathogens are small, and it is unable to cope with complex clinical infections. It is easy to miss detection; it is difficult to detect multiple pathogens and corresponding drug-resistant genes / mutations at the same time, and it is not easy to provide coherent diagnosis and medication guidance;

[0004] To better meet clinical testing needs, high-throughput sequencing technologies targeting pathogens of reproductive tract infections have emerged. Targeted pathogen sequencing (tNGS) is a next-generation sequencing technology based on ultra-multiplexed PCR amplification (or targeted capture) and high-throughput sequencing. It does not rely on traditional microbial culture. Instead, it directly enriches dozens to hundreds of known pathogens and their virulence and / or resistance genes in clinical samples. High-throughput sequencing is then performed, followed by comparison and analysis with a database. The identified sequence information is used to identify the pathogens present in the sample. This technology can rapidly and objectively detect a large number of pathogens in clinical samples, providing a basis for clinical diagnosis. It is particularly advantageous for detecting low concentrations of pathogens, particularly their virulence and / or resistance genes.

[0005] Compared with the aforementioned traditional detection methods, this method offers numerous advantages and features: 1) It can detect hundreds of different pathogens. 2) It is fast, typically taking less than 24 hours from sample receipt to the final report. 3) It is low cost per pathogen. 4) It can simultaneously detect a large number of pathogens and drug-resistance genes, providing more consistent diagnosis and medication guidance.

[0006] However, current high-throughput sequencing technologies targeting pathogens mainly focus on human respiratory tract-related pathogen infections, and lack detection technologies for reproductive tract pathogens, especially primers suitable for comprehensive screening of reproductive tract pathogens. Summary of the Invention

[0007] To overcome the shortcomings of existing high-throughput sequencing technologies targeting pathogens, which have limited ability to detect reproductive tract pathogens, the present invention provides a primer set, a kit, and applications thereof for high-throughput sequencing of pathogens of reproductive tract infections. The present invention is implemented using the following technical solutions:

[0008] A primer set for high-throughput sequencing of pathogens of reproductive tract infections, comprising:

[0009] HPV16 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 253, and the rear primer sequence is shown in seq ID No. 254;

[0010] HPV18 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 255, and the rear primer sequence is shown in seq ID No. 256;

[0011] HPV26 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 257, and the rear primer sequence is shown in seq ID No. 258;

[0012] HPV31 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 259, and the rear primer sequence is shown in seq ID No. 260;

[0013] HPV33 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 261, and the rear primer sequence is shown in seq ID No. 262;

[0014] HPV35 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 263, and the rear primer sequence is shown in seq ID No. 264;

[0015] HPV39 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 265, and the rear primer sequence is shown in seq ID No. 266;

[0016] HPV45 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 267, and the rear primer sequence is shown in seq ID No. 268;

[0017] HPV51 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 269, and the rear primer sequence is shown in seq ID No. 2700;

[0018] HPV52 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 271, and the rear primer sequence is shown in seq ID No. 272;

[0019] HPV53 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 273, and the rear primer sequence is shown in seq ID No. 274;

[0020] HPV56 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 275, and the rear primer sequence is shown in seq ID No. 276;

[0021] HPV58 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 277, and the rear primer sequence is shown in seq ID No. 278;

[0022] HPV59 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 279, and the rear primer sequence is shown in seq ID No. 280;

[0023] HPV66 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 281, and the rear primer sequence is shown in seq ID No. 282;

[0024] HPV67 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 283, and the rear primer sequence is shown in seq ID No. 284;

[0025] HPV68 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 285, and the rear primer sequence is shown in seq ID No. 286;

[0026] HPV73 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 287, and the rear primer sequence is shown in seq ID No. 288;

[0027] HPV82 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 289, and the rear primer sequence is shown in seq ID No. 290.

[0028] Optionally, at least one of the following detection primers is also included:

[0029] HPV6 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 291, and the rear primer sequence is shown in seq ID No. 292;

[0030] HPV11 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 293, and the rear primer sequence is shown in seq ID No. 294;

[0031] HPV40 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 295, and the rear primer sequence is shown in seq ID No. 296;

[0032] HPV42 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 297, and the rear primer sequence is shown in seq ID No. 298;

[0033] HPV43 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 299, and the rear primer sequence is shown in seq ID No. 300;

[0034] HPV54 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 301, and the rear primer sequence is shown in seq ID No. 302;

[0035] HPV61 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 303, and the rear primer sequence is shown in seq ID No. 304;

[0036] HPV70 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 305, and the rear primer sequence is shown in seq ID No. 306;

[0037] HPV72 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 307, and the rear primer sequence is shown in seq ID No. 308;

[0038] HPV81 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 309, and the rear primer sequence is shown in seq ID No. 310;

[0039] HPV89 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 311, and the rear primer sequence is shown in seq ID No. 312;

[0040] HPV34 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 313, and the rear primer sequence is shown in seq ID No. 314;

[0041] HPV44 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 315, and the rear primer sequence is shown in seq ID No. 316;

[0042] HPV57 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 317, and the rear primer sequence is shown in seq ID No. 318;

[0043] HPV69 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 319, and the rear primer sequence is shown in seq ID No. 320;

[0044] HPV71 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 321, and the rear primer sequence is shown in seq ID No. 322;

[0045] HPV83 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 323, and the rear primer sequence is shown in seq ID No. 324;

[0046] HPV84 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 325, and the rear primer sequence is shown in seq ID No. 326.

[0047] Optionally, it also includes at least one of primers for detecting beneficial symbiotic bacteria, primers for detecting anaerobic / facultative anaerobic bacteria, primers for detecting aerobic bacteria, primers for detecting STDs bacteria, primers for detecting mycobacteria, primers for detecting high-risk / suspected high-risk human papillomavirus, primers for detecting low-risk human papillomavirus, primers for detecting other DNA viruses, primers for detecting RNA viruses, primers for detecting fungi, and primers for detecting parasites; and / or

[0048] The beneficial symbiotic bacteria detection primers include:

[0049] Primers for detecting Lactobacillus crispatus, the forward primer sequence is shown in seq ID No. 1, and the rear primer sequence is shown in seq ID No. 2;

[0050] The primers for detecting Lactobacillus iners, the forward primer sequence is shown in seq ID No. 3, and the rear primer sequence is shown in seq ID No. 4;

[0051] The primers for detecting Lactobacillus jensenii are shown in seq ID No. 5 for the front primer and seq ID No. 6 for the back primer.

[0052] Lactobacillus gasseri detection primers, the forward primer sequence is shown in seq ID No. 7, and the rear primer sequence is shown in seq ID No. 8;

[0053] Lactobacillus detection primers, the forward primer sequence is shown in seq ID No. 9, and the rear primer sequence is shown in seq ID No. 10;

[0054] The anaerobic / facultative anaerobic bacteria detection primers include:

[0055] Primers for detecting Gardnerella vaginalis, the forward primer sequence is shown in seq ID No. 11, and the rear primer sequence is shown in seq ID No. 12;

[0056] The primers for detecting Atopobium vaginalis, the forward primer sequence is shown in seq ID No. 13, and the rear primer sequence is shown in seq ID No. 14;

[0057] The primers for detecting Bacteroides fragilis are shown in seq ID No. 19 for the forward primer and seq ID No. 20 for the rear primer.

[0058] Two-way Prevotella detection primers, the forward primer sequence is shown in seq ID No. 21, and the rear primer sequence is shown in seq ID No. 22;

[0059] Proteus mirabilis detection primers, the forward primer sequence is shown in seq ID No. 23, and the rear primer sequence is shown in seq ID No. 24;

[0060] Streptococcus agalactiae detection primers, the forward primer sequence is shown in seq ID No. 25, and the rear primer sequence is shown in seq ID No. 26;

[0061] Streptococcus pneumoniae detection primers, the forward primer sequence is shown in seq ID No. 27, and the rear primer sequence is shown in seq ID No. 28;

[0062] Anaerobic Peptostreptococcus detection primers, the forward primer sequence is shown in seq ID No. 29, and the rear primer sequence is shown in seq ID No. 30;

[0063] Primers for detecting F. magna, the forward primer sequence is shown in seq ID No. 31, and the rear primer sequence is shown in seq ID No. 32;

[0064] Micromonas pumila detection primers, the forward primer sequence is shown in seq ID No. 33, and the rear primer sequence is shown in seq ID No. 34;

[0065] The primers for detecting Clostridium perfringens, the forward primer sequence is shown in seq ID No. 35, and the rear primer sequence is shown in seq ID No. 36;

[0066] Mobiluncus timidus detection primers, the forward primer sequence is shown in seq ID No. 37, and the rear primer sequence is shown in seq ID No. 38;

[0067] Mobiluncus krusei detection primers, the forward primer sequence is shown in seq ID No. 39, and the rear primer sequence is shown in seq ID No. 40;

[0068] The primers for detecting Megasphaera are shown in seq ID No. 41 for the forward primer and seq ID No. 42 for the rear primer.

[0069] Actinomyces ilaudid detection primers: the forward primer sequence is shown in seq ID No. 43, and the rear primer sequence is shown in seq ID No. 44;

[0070] Oral Trichoderma detection primers, the forward primer sequence is shown in seq ID No. 45, and the rear primer sequence is shown in seq ID No. 46;

[0071] The primers for detecting Megasphaera elsdenii are shown in seq ID No. 47 for the forward primer and seq ID No. 48 for the rear primer.

[0072] Primers for aerobic bacteria detection include:

[0073] Staphylococcus aureus detection primers, the forward primer sequence is shown in seq ID No. 49, and the rear primer sequence is shown in seq ID No. 50;

[0074] Staphylococcus epidermidis detection primers, the forward primer sequence is shown in seq ID No. 51, and the rear primer sequence is shown in seq ID No. 52;

[0075] Staphylococcus intermedius detection primers, the forward primer sequence is shown in seq ID No. 53, and the rear primer sequence is shown in seq ID No. 54;

[0076] Primers for detecting hemolytic Staphylococcus aureus, the forward primer sequence is shown in seq ID No. 55, and the rear primer sequence is shown in seq ID No. 56;

[0077] Primers for Staphylococcus lugdunensis detection, the forward primer sequence is shown in seq ID No. 57, and the rear primer sequence is shown in seq ID No. 58;

[0078] Enterococcus faecalis detection primers, the forward primer sequence is shown in seq ID No. 59, and the rear primer sequence is shown in seq ID No. 60;

[0079] Enterococcus faecium detection primers, the forward primer sequence is shown in seq ID No. 61, and the rear primer sequence is shown in seq ID No. 62;

[0080] Escherichia coli detection primers, the forward primer sequence is shown in seq ID No. 63, and the rear primer sequence is shown in seq ID No. 64;

[0081] Klebsiella pneumoniae detection primers, the forward primer sequence is shown in seq ID No. 65, and the rear primer sequence is shown in seq ID No. 66;

[0082] Klebsiella oxytoca detection primers, the forward primer sequence is shown in seq ID No. 67, and the rear primer sequence is shown in seq ID No. 68;

[0083] Klebsiella aerogenes detection primers, the forward primer sequence is shown in seq ID No. 69, and the rear primer sequence is shown in seq ID No. 70;

[0084] Enterobacter cloacae complex detection primers, the forward primer sequence is shown in seq ID No. 71, and the rear primer sequence is shown in seq ID No. 72;

[0085] Primers for detecting Citrobacter cohnii, the forward primer sequence is shown in seq ID No. 73, and the rear primer sequence is shown in seq ID No. 74;

[0086] Primers for detecting Citrobacter freundii: the forward primer sequence is shown in seq ID No. 75, and the rear primer sequence is shown in seq ID No. 76;

[0087] Pseudomonas aeruginosa detection primers, the forward primer sequence is shown in seq ID No. 79, and the rear primer sequence is shown in seq ID No. 80;

[0088] Haemophilus influenzae detection primers, the forward primer sequence is shown in seq ID No. 81, and the rear primer sequence is shown in seq ID No. 82;

[0089] Haemophilus parainfluenzae detection primers, the forward primer sequence is shown in seq ID No. 83, and the rear primer sequence is shown in seq ID No. 84;

[0090] Streptococcus anginosus detection primers, the forward primer sequence is shown in seq ID No. 85, and the rear primer sequence is shown in seq ID No. 86;

[0091] STDs bacterial detection primers include:

[0092] Neisseria gonorrhoeae detection primers, the forward primer sequence is shown in seq ID No. 87, and the rear primer sequence is shown in seq ID No. 88;

[0093] Treponema pallidum detection primers, the forward primer sequence is shown in seq ID No. 89, and the rear primer sequence is shown in seq ID No. 90;

[0094] Haemophilus ducreyi detection primers, the forward primer sequence is shown in seq ID No. 91, and the rear primer sequence is shown in seq ID No. 92;

[0095] Chlamydia trachomatis detection primers, the forward primer sequence is shown in seq ID No. 93, and the rear primer sequence is shown in seq ID No. 94;

[0096] Mycoplasma hominis detection primers, the forward primer sequence is shown in seq ID No. 95, and the rear primer sequence is shown in seq ID No. 96;

[0097] Ureaplasma urealyticum detection primers, the forward primer sequence is shown in seq ID No. 97, and the rear primer sequence is shown in seq ID No. 98;

[0098] Mycoplasma genitalium detection primers, the forward primer sequence is shown in seq ID No. 99, and the rear primer sequence is shown in seq ID No. 100;

[0099] Mycobacterium detection primers include: Mycobacterium tuberculosis complex detection primers, the forward primer sequence is shown in seq ID No. 103, and the rear primer sequence is shown in seq ID No. 104;

[0100] High-risk and suspected high-risk human papillomavirus detection primers:

[0101] HPV16 detection primers, the forward primer sequence is shown in seq ID No. 135, and the rear primer sequence is shown in seq ID No. 136;

[0102] HPV18 detection primers, the forward primer sequence is shown in seq ID No. 137, and the rear primer sequence is shown in seq ID No. 138;

[0103] HPV31 detection primers, the forward primer sequence is shown in seq ID No. 139, and the rear primer sequence is shown in seq ID No. 140;

[0104] HPV33 detection primers, the forward primer sequence is shown in seq ID No. 141, and the rear primer sequence is shown in seq ID No. 142;

[0105] HPV35 detection primers, the forward primer sequence is shown in seq ID No. 143, and the rear primer sequence is shown in seq ID No. 144;

[0106] HPV39 detection primers, the forward primer sequence is shown in seq ID No. 145, and the rear primer sequence is shown in seq ID No. 146;

[0107] HPV45 detection primers, the forward primer sequence is shown in seq ID No. 147, and the rear primer sequence is shown in seq ID No. 148;

[0108] HPV51 detection primers, the forward primer sequence is shown in seq ID No. 149, and the rear primer sequence is shown in seq ID No. 150;

[0109] HPV52 detection primers, the forward primer sequence is shown in seq ID No. 151, and the rear primer sequence is shown in seq ID No. 152;

[0110] HPV56 detection primers, the forward primer sequence is shown in seq ID No. 153, and the rear primer sequence is shown in seq ID No. 154;

[0111] HPV58 detection primers, the forward primer sequence is shown in seq ID No. 155, and the rear primer sequence is shown in seq ID No. 156;

[0112] HPV59 detection primers, the forward primer sequence is shown in seq ID No. 157, and the rear primer sequence is shown in seq ID No. 158;

[0113] HPV66 detection primers, the forward primer sequence is shown in seq ID No. 159, and the rear primer sequence is shown in seq ID No. 160;

[0114] HPV68 detection primers, the forward primer sequence is shown in seq ID No. 161, and the rear primer sequence is shown in seq ID No. 162;

[0115] HPV26 detection primers, the forward primer sequence is shown in seq ID No. 163, and the rear primer sequence is shown in seq ID No. 164;

[0116] HPV53 detection primers, the forward primer sequence is shown in seq ID No. 165, and the rear primer sequence is shown in seq ID No. 166;

[0117] HPV67 detection primers, the forward primer sequence is shown in seq ID No. 167, and the rear primer sequence is shown in seq ID No. 168;

[0118] HPV70 detection primers, the forward primer sequence is shown in seq ID No. 169, and the rear primer sequence is shown in seq ID No. 170;

[0119] HPV73 detection primers, the forward primer sequence is shown in seq ID No. 171, and the rear primer sequence is shown in seq ID No. 172;

[0120] HPV82 detection primers, the forward primer sequence is shown in seq ID No. 173, and the rear primer sequence is shown in seq ID No. 174;

[0121] Low-risk human papillomavirus detection primers include:

[0122] HPV6 detection primers, the forward primer sequence is shown in seq ID No. 175, and the rear primer sequence is shown in seq ID No. 176;

[0123] HPV11 detection primers, the forward primer sequence is shown in seq ID No. 177, and the rear primer sequence is shown in seq ID No. 178;

[0124] HPV40 detection primers, the forward primer sequence is shown in seq ID No. 179, and the rear primer sequence is shown in seq ID No. 180;

[0125] HPV42 detection primers, the forward primer sequence is shown in seq ID No. 181, and the rear primer sequence is shown in seq ID No. 182;

[0126] HPV43 detection primers, the forward primer sequence is shown in seq ID No. 183, and the rear primer sequence is shown in seq ID No. 184;

[0127] HPV54 detection primers, the forward primer sequence is shown in seq ID No. 185, and the rear primer sequence is shown in seq ID No. 186;

[0128] HPV61 detection primers, the forward primer sequence is shown in seq ID No. 187, and the rear primer sequence is shown in seq ID No. 188;

[0129] HPV72 detection primers, the forward primer sequence is shown in seq ID No. 189, and the rear primer sequence is shown in seq ID No. 190;

[0130] HPV81 detection primers, the forward primer sequence is shown in seq ID No. 191, and the rear primer sequence is shown in seq ID No. 192;

[0131] HPV89 detection primers, the forward primer sequence is shown in seq ID No. 193, and the rear primer sequence is shown in seq ID No. 194;

[0132] Other DNA virus detection primers include:

[0133] Human herpesvirus type 1 detection primers, the forward primer sequence is shown in seq ID No. 209, and the rear primer sequence is shown in seq ID No. 210;

[0134] Human herpesvirus type II detection primers, the forward primer sequence is shown in seq ID No. 211, and the rear primer sequence is shown in seq ID No. 212;

[0135] Epstein-Barr virus detection primers, the forward primer sequence is shown in seq ID No. 213, and the rear primer sequence is shown in seq ID No. 214;

[0136] Cytomegalovirus detection primers, the forward primer sequence is shown in seq ID No. 215, and the rear primer sequence is shown in seq ID No. 216;

[0137] Molluscum contagiosum virus detection primers, the forward primer sequence is shown in seq ID No. 217, and the rear primer sequence is shown in seq ID No. 218;

[0138] Hepatitis B virus detection primers, the forward primer sequence is shown in seq ID No. 219, and the rear primer sequence is shown in seq ID No. 220;

[0139] Human polyomavirus type 1 detection primers, the forward primer sequence is shown in seq ID No. 221, and the rear primer sequence is shown in seq ID No. 222;

[0140] Human polyomavirus type 2 detection primers, the forward primer sequence is shown in seq ID No. 223, and the rear primer sequence is shown in seq ID No. 224;

[0141] Varicella-zoster virus detection primers, the forward primer sequence is shown in seq ID No. 225, and the rear primer sequence is shown in seq ID No. 226;

[0142] RNA virus detection primers include:

[0143] Human immunodeficiency virus type I detection primers, the forward primer sequence is shown in seq ID No. 125, and the rear primer sequence is shown in seq ID No. 126;

[0144] Human immunodeficiency virus type II detection primers, the forward primer sequence is shown in seq ID No. 127, and the rear primer sequence is shown in seq ID No. 128;

[0145] Hepatitis A virus detection primers, the forward primer sequence is shown in seq ID No. 129, and the rear primer sequence is shown in seq ID No. 130;

[0146] Hepatitis C virus detection primers, the forward primer sequence is shown in seq ID No. 131, and the rear primer sequence is shown in seq ID No. 132;

[0147] Human T-lymphotropic virus type 1 detection primers, the forward primer sequence is shown in seq ID No. 133, and the rear primer sequence is shown in seq ID No. 134;

[0148] Fungal detection primers include:

[0149] Candida albicans detection primers, the forward primer sequence is shown in seq ID No. 105, and the rear primer sequence is shown in seq ID No. 106;

[0150] Candida tropicalis detection primers, the forward primer sequence is shown in seq ID No. 107, and the rear primer sequence is shown in seq ID No. 108;

[0151] Candida glabrata detection primers, the forward primer sequence is shown in seq ID No. 109, and the rear primer sequence is shown in seq ID No. 110;

[0152] Candida krusei detection primers, the forward primer sequence is shown in seq ID No. 111, and the rear primer sequence is shown in seq ID No. 112;

[0153] Primers for detecting Candida albicans: the forward primer sequence is shown in seq ID No. 113, and the rear primer sequence is shown in seq ID No. 114;

[0154] Candida parapsilosis detection primers, the forward primer sequence is shown in seq ID No. 115, and the rear primer sequence is shown in seq ID No. 116;

[0155] Candida dunovica detection primers, the forward primer sequence is shown in seq ID No. 117, and the rear primer sequence is shown in seq ID No. 118;

[0156] Parasite detection primers include:

[0157] Trichomonas vaginalis detection primers, the forward primer sequence is shown in seq ID No. 119, and the rear primer sequence is shown in seq ID No. 120;

[0158] Entamoeba histolytica detection primers: the forward primer sequence is shown in seq ID No. 121, and the rear primer sequence is shown in seq ID No. 122;

[0159] Toxoplasma gondii detection primers: the forward primer sequence is shown in seq ID No. 123, and the rear primer sequence is shown in seq ID No. 124.

[0160] Optionally, also include:

[0161] The detection primers for the drug resistance gene 23SrRNA, the forward primer sequence is shown in seq ID No. 227, and the rear primer sequence is shown in seq ID No. 228;

[0162] The detection primers for the drug resistance gene parC, the forward primer sequence is shown in seq ID No. 229, and the rear primer sequence is shown in seq ID No. 230;

[0163] The detection primers for the drug resistance gene 16SrRNA, the forward primer sequence is shown in seq ID No. 231, and the rear primer sequence is shown in seq ID No. 231;

[0164] The detection primers for the drug resistance gene penA, the forward primer sequence is shown in seq ID No. 233, and the rear primer sequence is shown in seq ID No. 234;

[0165] The detection primers for the drug resistance gene ftsX, the forward primer sequence is shown in seq ID No. 235, and the rear primer sequence is shown in seq ID No. 236;

[0166] The detection primers for the drug resistance gene PBP2x, the forward primer sequence is shown in seq ID No. 237, and the rear primer sequence is shown in seq ID No. 238;

[0167] The detection primers for the drug resistance gene gyrA, the forward primer sequence is shown in seq ID No. 239, and the rear primer sequence is shown in seq ID No. 240;

[0168] The detection primers for the drug resistance gene grlA, the forward primer sequence is shown in seq ID No. 241, and the rear primer sequence is shown in seq ID No. 242;

[0169] The detection primers for the drug-resistant gene graS, the forward primer sequence is shown in seq ID No. 243, and the rear primer sequence is shown in seq ID No. 244;

[0170] The detection primers for the drug resistance gene rpoB, the forward primer sequence is shown in seq ID No. 245, and the rear primer sequence is shown in seq ID No. 246;

[0171] The detection primers for the drug resistance gene ntr6, the forward primer sequence is shown in seq ID No. 247, and the rear primer sequence is shown in seq ID No. 248;

[0172] The detection primers for the drug-resistant gene ERG11, the forward primer sequence is shown in seq ID No. 249, and the rear primer sequence is shown in seq ID No. 250.

[0173] Optionally, at least one of the following pathogen detection primers is also included:

[0174] Prevotella detection primers, the forward primer sequence is shown in seq ID No. 15, and the rear primer sequence is shown in seq ID No. 16;

[0175] Enterobacter hallii detection primers, the forward primer sequence is shown in seq ID No. 17, and the rear primer sequence is shown in seq ID No. 18;

[0176] Pseudomonas detection primers, the forward primer sequence is shown in seq ID No. 77, and the rear primer sequence is shown in seq ID No. 78;

[0177] HPV detection primers:

[0178] HPV34 detection primers, the forward primer sequence is shown in seq ID No. 195, and the rear primer sequence is shown in seq ID No. 196;

[0179] HPV44 detection primers, the forward primer sequence is shown in seq ID No. 197, and the rear primer sequence is shown in seq ID No. 198;

[0180] HPV57 detection primers, the forward primer sequence is shown in seq ID No. 199, and the rear primer sequence is shown in seq ID No. 200;

[0181] HPV69 detection primers, the forward primer sequence is shown in seq ID No. 201, and the rear primer sequence is shown in seq ID No. 202;

[0182] HPV71 detection primers, the forward primer sequence is shown in seq ID No. 203, and the rear primer sequence is shown in seq ID No. 204;

[0183] HPV83 detection primers, the forward primer sequence is shown in seq ID No. 205, and the rear primer sequence is shown in seq ID No. 206;

[0184] HPV84 detection primers, the forward primer sequence is shown in seq ID No. 207, and the rear primer sequence is shown in seq ID No. 208.

[0185] Optionally, an internal standard sequence is also included, and the nucleic acid sequence of the internal standard sequence is shown as seq ID No.251 as the front primer sequence, and the rear primer sequence is shown as seq ID No.252 as the back primer sequence.

[0186] Optionally, the detection primers are all connected to a linker sequence; the nucleic acid sequence of the linker sequence is shown as seq ID No.327 for the front primer and as seq ID No.328 for the rear primer.

[0187] A kit comprising the above-mentioned primer set for high-throughput sequencing targeting reproductive tract infection pathogens.

[0188] Optionally, the kit further comprises a gDNA digestion reaction reagent, a single-strand synthesis reaction reagent, a first-step amplification reaction reagent, and a second-step amplification reaction reagent;

[0189] The reagent components of the gDNA digestion reaction include: gDNA wiper Mix reagent;

[0190] The reagent components of the single-strand synthesis reaction include: RT Mix reagent, Enzyme Mix reagent, nuclease-free water and Random Primers reagent;

[0191] The reagent components of the first step amplification reaction include: reaction buffer, nuclease-free water, multiplex amplification enzyme and PIC;

[0192] The reagent components of the second step amplification reaction include: Hifi Mix reagent.

[0193] The primer set and the kit for high-throughput sequencing targeting pathogens of reproductive tract infections are used for high-throughput sequencing to detect pathogens in reproductive tract infection samples.

[0194] Compared with the prior art, the present invention has the following beneficial effects:

[0195] The present invention can detect HPV E6 / E7 mRNA, that is, detect whether the HPV virus is persistently infecting the patient's body, and provide more accurate clinical triage guidance.

[0196] The detection scope includes common microorganisms in gynecological reproductive tract infections in a relatively detailed manner, and accurately classifies them into "pathogenic microorganisms", "suspected human microecological flora", and "beneficial symbiotic bacteria" based on their degree of harm, which helps to achieve accurate diagnosis; the application of this detection technology in the detection of pathogens and drug-resistant genes related to reproductive tract infections has important clinical significance, greatly increasing the number of detected pathogens and drug-resistant genes in a short period of time and reducing detection costs.

[0197] Based on the above primers, multiplex PCR technology and high-throughput sequencing methods are used to detect pathogens and drug-resistant genes related to reproductive tract infections. Gene fragments of specific detection areas of multiple drug-resistant genes and pathogens are amplified simultaneously. The detection capability is strong, the sensitivity is high, the detection range is wide, the speed is fast, and the average detection cost of a single gene or pathogen is low. DETAILED DESCRIPTION

[0198] The present invention is further described in detail below with reference to specific embodiments. The following embodiments are provided for a better understanding of the present invention, but are not limited to the best mode of implementation, and do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0199] If specific experimental procedures or conditions are not specified in the examples, the experiments were performed according to the conventional experimental procedures or conditions described in the literature in the field. Reagents or instruments used without manufacturer's indication are commercially available. Raw materials used unless otherwise specified are commercially available products that are readily available to those skilled in the art.

[0200] Example 1

[0201] Design and obtain primers. The primers required to be designed in this embodiment are primers for detecting E6 / E7 mRNA of HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV67, HPV68, HPV73, and HPV82.

[0202] The primer design method refers to Chinese patent ZL202411253978.9 and includes the following steps: Step 1) In-depth understanding of the biological characteristics of pathogens or specific gene regions related to the reproductive tract, obtaining genomic data of pathogenic microorganisms, and establishing a pathogenic microorganism genome database.

[0203] Step 2) Compare the homology and differences between different species or strains, and obtain the conserved regions of pathogenic microorganisms based on the pathogenic microorganism genome database.

[0204] Step 3) Design multiple primers based on the conserved regions from Step 2), taking into account parameters such as primer length, GC content, and Tm value. Generally, primers are typically 18-25 bases in length with a moderate GC content to ensure stability and specificity. Tm values should be similar to facilitate amplification under the same reaction conditions. Furthermore, the formation of secondary structures within the primers and complementarity between primers should be avoided.

[0205] Step 4) Evaluate the specificity and inclusiveness of the multiplex primers designed in Step 3) and select those with specificity and inclusiveness ≥ 90%. If any issues are identified, readjust the primer design or further optimize the experimental conditions to ensure that the genital tract-targeted primers can be accurately and efficiently used for genital tract-related testing and research.

[0206] The primers obtained are shown in Table 3. The primer set composed of the above primers can meet the detection requirements of HPV E6 / E7 mRNA under general circumstances.

[0207] Example 2

[0208] In this example, based on the primer set of Example 1, primers for detecting E6 / E7 mRNA of HPV6, HPV11, HPV40, HPV42, HPV43, HPV54, HPV61, HPV70, HPV72, HPV81, HPV89, HPV34, HPV44, HPV57, HPV69, HPV71, HPV83, and HPV84 were further designed and added to the primer set of Example 1 to obtain a high-throughput sequencing primer set that can comprehensively detect HPV E6 / E7 mRNA. The obtained primers are specifically shown in Table 3.

[0209] Example 3

[0210] In this example, based on the primer set of Example 1, primers for detecting pathogens listed in Table 1 and primers for detecting drug-resistant genes listed in Table 2 were further designed and added to the primer set of Example 1. This primer set covers a relatively comprehensive range of microorganisms commonly found in gynecological reproductive tract infections and accurately classifies them into "pathogenic microorganisms," "suspected human microecological flora," and "beneficial symbiotic bacteria" based on their degree of harm. This helps achieve accurate diagnosis, significantly increases the number of pathogens and drug-resistant genes detected in a short period of time, and reduces testing costs.

[0211] Table 1 List of pathogenic microorganisms

[0212]

[0213] The designed drug-resistant gene detection primers in this embodiment include detection primers for the drug-resistant gene 23SrRNA, drug-resistant gene parC, drug-resistant gene 16SrRNA, drug-resistant gene penA, drug-resistant gene ftsX, drug-resistant gene PBP2x, drug-resistant gene gyrA, drug-resistant gene grlA, drug-resistant gene graS, drug-resistant gene rpoB, drug-resistant gene ntr6, and drug-resistant gene ERG11. The various drug-resistant genes and their corresponding pathogens and drug-resistant gene sites are shown in Table 2.

[0214] Table 2 Drug resistance gene loci

[0215]

[0216] The detection primers obtained in Examples 1 to 3 are shown in Table 3.

[0217] Table 3 Primer sequences

[0218]

[0219] Example 4

[0220] This embodiment provides a kit for detecting reproductive tract pathogens by high-throughput sequencing using the above-mentioned primer set. The kit comprises:

[0221] The primer combination, gDNA digestion reaction, single-strand synthesis reaction, first-step amplification reaction, and reagents for the second-step amplification reaction in Example 3.

[0222] The reagents for the gDNA digestion reaction are: 5× gDNA wiper mix, nuclease-free water; the reagents for the single-strand synthesis reaction are: 10×RT mix, 10× enzyme mix, random primers, nuclease-free water;

[0223] The reagents for the first step amplification reaction are: 5× Reaction buffer, multiplex amplification enzyme, and PIC;

[0224] The reagents for the second-step amplification reaction were: 2 × Hifi Mix, nuclease-free water, and adapters;

[0225] The gDNA wiper mix used nucleic acid extraction or purification reagents (96 servings) from Guangzhou Heyuan Biotechnology Co., Ltd. The RT mix, enzyme mix, and random primers were from Guangzhou Dayuanqi Biotechnology Co., Ltd.'s RNA single-strand synthesis reagent-3 (96 servings). The reaction buffer, multiplex amplification enzyme, PIC, HiFi mix, and adapters were from Guangzhou Dayuanqi Biotechnology Co., Ltd.'s multiplex library buffer-2 (96 servings). Also used in this example were MagPure A3 XP1# (purification magnetic beads), tNGS supporting reagents, a reproductive tract pathogen detection panel (MGI platform), and MGI platform library construction index primers (1-24), all from Guangzhou Dayuanqi Biotechnology Co., Ltd.

[0226] Hifi Mix, adapters, PIC, etc., RNA single-strand synthesis reagent-3 from Guangzhou Dayuanqi Biotechnology Co., Ltd., multiple reconstruction buffer-2, MagPure A3 XP1# (purification magnetic beads), tNGS supporting reagents, reproductive tract pathogen detection panel (MGI platform), MGI platform library construction index primers (1-24).

[0227] The reaction system for the gDNA digestion reaction is 10 μL, and its composition is as follows: 2 μL of 5× gDNA wiper mix, no more than 400 ng of template, and nuclease-free water to make up the volume to 10 μL;

[0228] The reaction system for the first-strand synthesis reaction is 20 μL, and its composition is as follows: 10 μL of gDNA digestion product, 2 μL of 10×RTMix, 2 μL of 10×Enzyme Mix, 1 μL of Random Primers, and 5 μL of nuclease-free water;

[0229] The first step amplification reaction system is divided into a DNA amplification system and an RNA amplification system. The DNA amplification system is 10 μL and is composed of the following: 2 μL of 5 × Reaction buffer, 2 μL of multiplex PCR primers, 0.5 μL of multiplex amplification enzyme, 0.5 μL of PIC, and 5 μL of nucleic acid extraction product; the RNA amplification system is 10 μL and is composed of the following: 2 μL of 5 × Reaction buffer, 2 μL of multiplex PCR primers, 0.5 μL of multiplex amplification enzyme, 0.5 μL of PIC, and 5 μL of cDNA first-strand product.

[0230] The second step amplification reaction system is 50 μL, and its composition is as follows: 2× Hifi Mix 25 μL, adapter 5 μL, and purified product of the first amplification 20 μL;

[0231] The gDNA digestion reaction procedure was as follows: heated lid at 105°C, 42°C for 2 min, and incubation at 4°C;

[0232] The procedure for the one-strand synthesis reaction was as follows: heated lid at 105°C, 25°C for 5 min, 42°C for 20 min, 85°C for 5 min, and insulation at 4°C;

[0233] The program of the first step amplification reaction is as follows: 95°C for 10 min, 1 cycle; 95°C for 30 s, 61°C for 2 min, 63°C for 3 min, 65°C for 1 min, 72°C for 1 min, 5 cycles; 95°C for 30 s, 70°C for 3 min, 25 cycles; incubation at 4°C;

[0234] The program of the second step amplification reaction is: 98°C 45s, 1 cycle; 98°C 15s, 60°C 30s, 72°C 30s, 6 cycles; 72°C 5min; and insulation at 4°C.

[0235] The reagents for the gDNA digestion reaction include 5× gDNA wiper mix; the reagents for the first-strand synthesis reaction include 10×RTMix, 10×Enzyme Mix, and Random Primers; the reagents for the first-step amplification reaction system include 5× Reaction buffer, multiplex PCR primers, multiplex amplification enzyme, and PIC; the reagents for the second-step amplification reaction system include 2× HiFi Mix and adapters.

[0236] A method for high-throughput sequencing of targeted reproductive tract infection pathogens based on the above-mentioned kit comprises the following steps:

[0237] 1. Co-extraction of pathogen DNA and RNA

[0238] 1) Take a clean, broken tube (MP Lysing Matrix E tube) and add 1000 μL of lysis buffer.

[0239] 2) Vortex the sample and transfer 400 µL of the sample into a cell wall breaking tube. Fragment the cell wall using a cell wall breaking instrument (program: vibration intensity (SPEED) M / S = 6.0, run time 30 s, pause time 30 s, number of cycles 8).

[0240] 3) Centrifuge at high speed, 12000 rcf, 1 min, and transfer the supernatant (about 1 mL) to a new 2.0 mL tube for extraction.

[0241] 4) Add 30 μL of extraction magnetic beads and 40 μL of proteinase K to the 2 ml centrifuge tube containing the sample and vortex to mix.

[0242] 5) Place in a 70°C constant temperature shaking metal bath at 1200 rpm for 10 min. Immediately spin down after incubation.

[0243] 6) Place the centrifuge tube on the magnetic stand and carefully remove the liquid with a pipette when the magnetic beads are completely adsorbed.

[0244] 7) Add 800 μL of washing solution, vortex mix for 1 min, and centrifuge.

[0245] 8) Place the centrifuge tube on the magnetic stand and carefully remove the liquid with a pipette when the magnetic beads are completely adsorbed.

[0246] 9) Add 800 μL of washing solution, vortex mix for 1 min, and centrifuge.

[0247] 10) Place the centrifuge tube on the magnetic rack. When the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette and repeat this process.

[0248] 11) Centrifuge for 10 seconds and place the tube back into the magnetic rack. After all the magnetic beads are absorbed, use a 10μL pipette to remove the liquid at the bottom of the tube. Place the centrifuge tube on the magnetic rack and let it dry at room temperature.

[0249] 12) Add 50 μL of elution buffer, vortex to mix, and centrifuge briefly. Incubate at room temperature for 5 minutes, and then place on a magnetic rack.

[0250] 13) When the magnetic beads are completely adsorbed, transfer the nucleic acid solution to a new 1.5 mL EP tube.

[0251] 2. gDNA Digestion

[0252] The pathogen DNA and RNA co-extraction products were shaken and mixed, and then centrifuged instantaneously.

[0253] Measure the DNA concentration of the co-extraction product of pathogen DNA and RNA, and calculate the volume of "total nucleic acid" and "nuclease-free water" required in Table 4.

[0254] Prepare the gDNA digestion reaction system, mix gently, and centrifuge briefly. The formula is shown in Table 4:

[0255] Table 4 gDNA digestion reaction system

[0256]

[0257] (4) Place the reaction system into a conventional PCR instrument. The reaction program is shown in Table 5:

[0258] Table 5 gDNA digestion system reaction procedure

[0259]

[0260] 3. Single-strand synthesis

[0261] Vortex and mix the gDNA digestion product, and centrifuge briefly.

[0262] Prepare the first-strand synthesis reaction system, mix gently, and centrifuge briefly. The formula is shown in Table 6:

[0263] Table 6 One-chain synthesis reaction system

[0264]

[0265] Place the single-strand synthesis reaction system into a conventional PCR instrument, and the reaction program is shown in Table 7:

[0266] Table 7 Reaction procedures for one-chain synthesis system

[0267]

[0268] Note: cDNA can be used immediately in multiple amplification experiments. Avoid repeated freezing and thawing of cDNA. For short-term storage, it is recommended to store at -20°C; for long-term storage, it is recommended to store at -70°C.

[0269] 4. First step PCR amplification

[0270] This detection method primarily involves designing multiplex PCR-specific primers based on specific regions of pathogens and drug-resistance genes within the detection range. PCR amplification is then performed, and the amplified products are purified and recovered before high-throughput sequencing. The primers in the primer set are not modified, and the formulation and ratio of the PCR reagents ensure consistent PCR amplification conditions across different gene loci, resulting in no nonspecific amplification. The primers in the amplification primer combination used in the first PCR amplification step should be linked to a universal linker sequence. Details of the base sequences are shown in Table 8.

[0271] Table 8 Base sequences of universal adapter sequences for multiplex PCR primers

[0272]

[0273] The single-strand synthesis product, pathogen DNA and RNA co-extraction products were shaken and mixed, and then centrifuged instantaneously.

[0274] Prepare the first step of the PCR amplification synthesis reaction system. The amount of primers used in each reaction system is 2µL. The formulas of the remaining reagents in the system are shown in Tables 9 and 10:

[0275] Table 9 DNA amplification system

[0276]

[0277] Table 10 RNA amplification system

[0278]

[0279] Place the first step PCR amplification synthesis reaction system into a conventional PCR instrument, and the reaction program is shown in Table 11:

[0280] Table 11 First step PCR amplification reaction program

[0281]

[0282] 5. First step magnetic bead purification

[0283] 30 minutes before the first round of magnetic bead purification, place the purification magnetic beads at room temperature to allow them to equilibrate to room temperature.

[0284] Combine the first-round amplification products of DNA and RNA in one tube, add 30 μL of nuclease-free water, and then add 60 μL of DNA purification magnetic beads;

[0285] After mixing evenly, let it stand at room temperature for 5 minutes. After a brief centrifugation, place it on a magnetic stand and let it stand for 3-5 minutes to separate the magnetic beads.

[0286] When the supernatant is completely clear, remove it.

[0287] Add 200 μL of freshly prepared 80% ethanol to the remaining magnetic beads, let it stand at room temperature for 30 seconds, remove the supernatant, and repeat the 80% ethanol wash once.

[0288] After a brief centrifugation, place the beads on a magnetic rack to completely remove the remaining ethanol and allow them to air-dry at room temperature for 2-3 minutes.

[0289] Add 23 μL of nuclease-free water to resuspend the magnetic beads, place at room temperature for 2 minutes, centrifuge briefly, and place on a magnetic rack for 2 minutes to separate the magnetic beads.

[0290] Pipette 20 μL of supernatant into a new 200 μL PCR tube, make sure to mark it, and place the product at 4°C until use, and store it at -20°C for sample storage.

[0291] 6. Second step PCR amplification

[0292] The first round of magnetic bead purification of pathogens was shaken and mixed, and then centrifuged briefly.

[0293] Prepare the second step PCR amplification reaction system, mix gently, and centrifuge briefly. The formula is shown in Table 12:

[0294] Table 12 Second step PCR amplification system

[0295]

[0296] Place the second-step PCR amplification reaction system into a conventional PCR instrument, and the reaction program is shown in Table 13:

[0297] Table 13 First step PCR amplification reaction program

[0298]

[0299] 7. Second step magnetic bead purification

[0300] After the second step of PCR amplification, the reaction tube was centrifuged briefly, 50 μL of nuclease-free water was added, and 75 μL of DNA purification magnetic beads were added to the PCR product, mixed, and incubated at room temperature for 5 min.

[0301] Centrifuge briefly, place the eight-tube strip on a magnetic rack, and transfer the supernatant to a PCR tube containing 15 μL DNA purification magnetic beads after it is completely clear. Mix well and incubate at room temperature for 5 min.

[0302] Centrifuge briefly, place the eight-tube strip on a magnetic rack, discard the supernatant after it is completely clear, add 200 μL 80% ethanol, let it stand for 30 seconds, discard the supernatant, and repeat the 80% ethanol wash once.

[0303] Centrifuge for 5 seconds, place the eight-tube strip on a magnetic rack, and discard the remaining liquid.

[0304] Dry the magnetic beads, add 40 μL of nuclease-free water to resuspend the magnetic beads, mix well, and let it stand for 2 minutes.

[0305] After a brief centrifugation, place the eight-tube strip on a magnetic stand and transfer 38 μL of supernatant to a new low-adsorption tube.

[0306] The purified product was mixed by vortexing, centrifuged briefly, and then the DNA concentration was measured.

[0307] 8. Second step magnetic bead purification

[0308] After library amplification, the reaction tube was centrifuged briefly, 50 μL of nuclease-free water was added, and 75 μL of DNA purification magnetic beads were added to the PCR product, mixed, and incubated at room temperature for 5 min.

[0309] Centrifuge briefly, place on a magnetic rack, and transfer the supernatant to a PCR tube containing 15 μL DNA purification magnetic beads after it is completely clear. Mix well and incubate at room temperature for 5 min.

[0310] Centrifuge briefly, place on a magnetic stand, discard the supernatant after it is completely clear, add 200 μL 80% ethanol, let it stand for 30 seconds, discard the supernatant, and repeat the 80% ethanol wash once.

[0311] Centrifuge for 5 seconds, place on a magnetic stand, and discard the remaining liquid.

[0312] Dry the magnetic beads, add 40 μL of nuclease-free water to resuspend the magnetic beads, mix well, and let it stand for 2 minutes.

[0313] After brief centrifugation, place on a magnetic stand and transfer 38 μL of supernatant to a new low-binding tube.

[0314] 9. Measure concentration

[0315] Measure the DNA concentration of the second-step purification product.

[0316] 10. Library mixing and DNB reaction: First, mix the outgoing libraries, and then use the MGI DNB kit to perform DNB reaction to obtain DNB products that can be finally loaded onto the instrument.

[0317] 11. Sequencing on a high-throughput sequencer

[0318] The qualified end products of the DNB reaction were sequenced on the MGISEQ series sequencer manufactured by MGI according to the instructions.

[0319] Test Example 1

[0320] 1. Experimental Methods

[0321] The high-throughput sequencing method targeting reproductive tract infection pathogens in Example 4 was used to detect common vaginal Gardnerella, beneficial bacteria, suspected human microecological bacteria and drug-resistant genes within the detection range.

[0322] 2. Experimental Results

[0323] As shown in Table 14, the high-throughput sequencing method targeting reproductive tract infection pathogens in Example 4 can effectively detect Gardnerella vaginalis when testing a sample with a known positive test result for Gardnerella vaginalis.

[0324] Table 14 High-throughput detection results of reproductive tract infection pathogens

[0325]

[0326] Test Example 2

[0327] 1. Experimental Methods

[0328] Three clinical specimens of reproductive tract infection were tested using a high-throughput sequencing method targeting reproductive tract infection pathogens in Example 4, and the test results were then compared with the results of the metagenomic detection method.

[0329] 2. Experimental Results

[0330] The experimental results are shown in Table 15.

[0331] Table 15 Comparison of high-throughput sequencing and metagenomic detection results of pathogens targeting reproductive tract infections

[0332]

[0333] Test Example 3

[0334] 1. Experimental Methods

[0335] The high-throughput sequencing method targeting reproductive tract infection pathogens in Example 4 was used to detect three clinical specimens of reproductive tract infection. Real-time fluorescence PCR was used to qualitatively detect the E6 / E7 region mRNA of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) in human cervical exfoliated cells from the three clinical specimens of reproductive tract infection. The results of the high-throughput sequencing method were then compared with the results of the real-time fluorescence PCR method.

[0336] 2. Experimental Results

[0337] The experimental results are shown in Table 16.

[0338] Table 16 Comparison of results of high-throughput sequencing and real-time fluorescence PCR for E6 / E7 region mRNA detection of pathogens of reproductive tract infection

[0339]

[0340] Furthermore, it should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A primer set for high-throughput sequencing of pathogens of reproductive tract infections, characterized in that: include: HPV16 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 253, and the rear primer sequence is shown in seq ID No. 254; HPV18 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 255, and the rear primer sequence is shown in seq ID No. 256; HPV26 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 257, and the rear primer sequence is shown in seq ID No. 258; HPV31 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 259, and the rear primer sequence is shown in seq ID No. 260; HPV33 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 261, and the rear primer sequence is shown in seq ID No. 262; HPV35 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 263, and the rear primer sequence is shown in seq ID No. 264; HPV39 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 265, and the rear primer sequence is shown in seq ID No. 266; HPV45 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 267, and the rear primer sequence is shown in seq ID No. 268; HPV51 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 269, and the rear primer sequence is shown in seq ID No. 270; HPV52 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 271, and the rear primer sequence is shown in seq ID No. 272; HPV53 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 273, and the rear primer sequence is shown in seq ID No. 274; HPV56 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 275, and the rear primer sequence is shown in seq ID No. 276; HPV58 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 277, and the rear primer sequence is shown in seq ID No. 278; HPV59 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 279, and the rear primer sequence is shown in seq ID No. 280; HPV66 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 281, and the rear primer sequence is shown in seq ID No. 282; HPV67 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 283, and the rear primer sequence is shown in seq ID No. 284; HPV68 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 285, and the rear primer sequence is shown in seq ID No. 286; HPV73 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 287, and the rear primer sequence is shown in seq ID No. 288; HPV82 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 289, and the rear primer sequence is shown in seq ID No. 290; The primer set for high-throughput sequencing targeting reproductive tract infection pathogens also includes primers for detecting beneficial commensal bacteria, primers for detecting anaerobic / facultative anaerobic bacteria, primers for detecting aerobic bacteria, primers for detecting STDs bacteria, primers for detecting mycobacteria, primers for detecting high-risk / suspected high-risk human papillomavirus, primers for detecting low-risk human papillomavirus, primers for detecting other DNA viruses, primers for detecting RNA viruses, primers for detecting fungi, and primers for detecting parasites; The beneficial symbiotic bacteria detection primers include: Primers for detecting Lactobacillus crispatus, the forward primer sequence is shown in seq ID No. 1, and the rear primer sequence is shown in seq ID No. 2; The primers for detecting Lactobacillus iners, the forward primer sequence is shown in seq ID No. 3, and the rear primer sequence is shown in seq ID No. 4; The primers for detecting Lactobacillus jensenii are shown in seq ID No. 5 for the front primer and seq ID No. 6 for the back primer. Lactobacillus gasseri detection primers, the forward primer sequence is shown in seq ID No. 7, and the rear primer sequence is shown in seq ID No. 8; Lactobacillus detection primers, the forward primer sequence is shown in seq ID No. 9, and the rear primer sequence is shown in seq ID No. 10; The anaerobic / facultative anaerobic bacteria detection primers include: Primers for detecting Gardnerella vaginalis, the forward primer sequence is shown in seq ID No. 11, and the rear primer sequence is shown in seq ID No. 12; The primers for detecting Atopobium vaginalis, the forward primer sequence is shown in seq ID No. 13, and the rear primer sequence is shown in seq ID No. 14; The primers for detecting Bacteroides fragilis are shown in seq ID No. 19 for the forward primer and seq ID No. 20 for the rear primer. Two-way Prevotella detection primers, the forward primer sequence is shown in seq ID No. 21, and the rear primer sequence is shown in seq ID No. 22; Proteus mirabilis detection primers, the forward primer sequence is shown in seq ID No. 23, and the rear primer sequence is shown in seq ID No. 24; Streptococcus agalactiae detection primers, the forward primer sequence is shown in seq ID No. 25, and the rear primer sequence is shown in seq ID No. 26; Streptococcus pneumoniae detection primers, the forward primer sequence is shown in seq ID No. 27, and the rear primer sequence is shown in seq ID No. 28; Anaerobic Peptostreptococcus detection primers, the forward primer sequence is shown in seq ID No. 29, and the rear primer sequence is shown in seq ID No. 30; Primers for detecting F. magna, the forward primer sequence is shown in seq ID No. 31, and the rear primer sequence is shown in seq ID No. 32; Micromonas pumila detection primers, the forward primer sequence is shown in seq ID No. 33, and the rear primer sequence is shown in seq ID No. 34; The primers for detecting Clostridium perfringens, the forward primer sequence is shown in seq ID No. 35, and the rear primer sequence is shown in seq ID No. 36; Mobiluncus timidus detection primers, the forward primer sequence is shown in seq ID No. 37, and the rear primer sequence is shown in seq ID No. 38; Mobiluncus krusei detection primers, the forward primer sequence is shown in seq ID No. 39, and the rear primer sequence is shown in seq ID No. 40; Actinomyces ilaudid detection primers: the forward primer sequence is shown in seq ID No. 43, and the rear primer sequence is shown in seq ID No. 44; Oral Trichoderma detection primers, the forward primer sequence is shown in seq ID No. 45, and the rear primer sequence is shown in seq ID No. 46; The primers for detecting Megasphaera elsdenii are shown in seq ID No. 47 for the forward primer and seq ID No. 48 for the rear primer. The aerobic bacteria detection primers include: Staphylococcus aureus detection primers, the forward primer sequence is shown in seq ID No. 49, and the rear primer sequence is shown in seq ID No. 50; Staphylococcus epidermidis detection primers, the forward primer sequence is shown in seq ID No. 51, and the rear primer sequence is shown in seq ID No. 52; Staphylococcus intermedius detection primers, the forward primer sequence is shown in seq ID No. 53, and the rear primer sequence is shown in seq ID No. 54; Primers for detecting hemolytic Staphylococcus aureus, the forward primer sequence is shown in seq ID No. 55, and the rear primer sequence is shown in seq ID No. 56; Primers for Staphylococcus lugdunensis detection, the forward primer sequence is shown in seq ID No. 57, and the rear primer sequence is shown in seq ID No. 58; Enterococcus faecalis detection primers, the forward primer sequence is shown in seq ID No. 59, and the rear primer sequence is shown in seq ID No. 60; Enterococcus faecium detection primers, the forward primer sequence is shown in seq ID No. 61, and the rear primer sequence is shown in seq ID No. 62; Escherichia coli detection primers, the forward primer sequence is shown in seq ID No. 63, and the rear primer sequence is shown in seq ID No. 64; Klebsiella pneumoniae detection primers, the forward primer sequence is shown in seq ID No. 65, and the rear primer sequence is shown in seq ID No. 66; Klebsiella oxytoca detection primers, the forward primer sequence is shown in seq ID No. 67, and the rear primer sequence is shown in seq ID No. 68; Klebsiella aerogenes detection primers, the forward primer sequence is shown in seq ID No. 69, and the rear primer sequence is shown in seq ID No. 70; Enterobacter cloacae complex detection primers, the forward primer sequence is shown in seq ID No. 71, and the rear primer sequence is shown in seq ID No. 72; Primers for detecting Citrobacter cohnii, the forward primer sequence is shown in seq ID No. 73, and the rear primer sequence is shown in seq ID No. 74; Primers for detecting Citrobacter freundii: the forward primer sequence is shown in seq ID No. 75, and the rear primer sequence is shown in seq ID No. 76; Pseudomonas aeruginosa detection primers, the forward primer sequence is shown in seq ID No. 79, and the rear primer sequence is shown in seq ID No. 80; Haemophilus influenzae detection primers, the forward primer sequence is shown in seq ID No. 81, and the rear primer sequence is shown in seq ID No. 82; Haemophilus parainfluenzae detection primers, the forward primer sequence is shown in seq ID No. 83, and the rear primer sequence is shown in seq ID No. 84; Streptococcus anginosus detection primers, the forward primer sequence is shown in seq ID No. 85, and the rear primer sequence is shown in seq ID No. 86; The STDs bacteria detection primers include: Neisseria gonorrhoeae detection primers, the forward primer sequence is shown in seq ID No. 87, and the rear primer sequence is shown in seq ID No. 88; Treponema pallidum detection primers, the forward primer sequence is shown in seq ID No. 89, and the rear primer sequence is shown in seq ID No. 90; Haemophilus ducreyi detection primers, the forward primer sequence is shown in seq ID No. 91, and the rear primer sequence is shown in seq ID No. 92; Chlamydia trachomatis detection primers, the forward primer sequence is shown in seq ID No. 93, and the rear primer sequence is shown in seq ID No. 94; Mycoplasma hominis detection primers, the forward primer sequence is shown in seq ID No. 95, and the rear primer sequence is shown in seq ID No. 96; Ureaplasma urealyticum detection primers, the forward primer sequence is shown in seq ID No. 97, and the rear primer sequence is shown in seq ID No. 98; Mycoplasma genitalium detection primers, the forward primer sequence is shown in seq ID No. 99, and the rear primer sequence is shown in seq ID No. 100; The mycobacterium detection primers include: Mycobacterium tuberculosis complex detection primers, the front primer sequence is shown in seq ID No. 103, and the rear primer sequence is shown in seq ID No. 104; The high-risk / suspected high-risk human papillomavirus detection primers: HPV16 detection primers, the forward primer sequence is shown in seq ID No. 135, and the rear primer sequence is shown in seq ID No. 136; HPV18 detection primers, the forward primer sequence is shown in seq ID No. 137, and the rear primer sequence is shown in seq ID No. 138; HPV31 detection primers, the forward primer sequence is shown in seq ID No. 139, and the rear primer sequence is shown in seq ID No. 140; HPV33 detection primers, the forward primer sequence is shown in seq ID No. 141, and the rear primer sequence is shown in seq ID No. 142; HPV35 detection primers, the forward primer sequence is shown in seq ID No. 143, and the rear primer sequence is shown in seq ID No. 144; HPV39 detection primers, the forward primer sequence is shown in seq ID No. 145, and the rear primer sequence is shown in seq ID No. 146; HPV45 detection primers, the forward primer sequence is shown in seq ID No. 147, and the rear primer sequence is shown in seq ID No. 148; HPV51 detection primers, the forward primer sequence is shown in seq ID No. 149, and the rear primer sequence is shown in seq ID No. 150; HPV52 detection primers, the forward primer sequence is shown in seq ID No. 151, and the rear primer sequence is shown in seq ID No. 152; HPV56 detection primers, the forward primer sequence is shown in seq ID No. 153, and the rear primer sequence is shown in seq ID No. 154; HPV58 detection primers, the forward primer sequence is shown in seq ID No. 155, and the rear primer sequence is shown in seq ID No. 156; HPV59 detection primers, the forward primer sequence is shown in seq ID No. 157, and the rear primer sequence is shown in seq ID No. 158; HPV66 detection primers, the forward primer sequence is shown in seq ID No. 159, and the rear primer sequence is shown in seq ID No. 160; HPV68 detection primers, the forward primer sequence is shown in seq ID No. 161, and the rear primer sequence is shown in seq ID No. 162; HPV26 detection primers, the forward primer sequence is shown in seq ID No. 163, and the rear primer sequence is shown in seq ID No. 164; HPV53 detection primers, the forward primer sequence is shown in seq ID No. 165, and the rear primer sequence is shown in seq ID No. 166; HPV67 detection primers, the forward primer sequence is shown in seq ID No. 167, and the rear primer sequence is shown in seq ID No. 168; HPV70 detection primers, the forward primer sequence is shown in seq ID No. 169, and the rear primer sequence is shown in seq ID No. 170; HPV73 detection primers, the forward primer sequence is shown in seq ID No. 171, and the rear primer sequence is shown in seq ID No. 172; HPV82 detection primers, the forward primer sequence is shown in seq ID No. 173, and the rear primer sequence is shown in seq ID No. 174; The low-risk human papillomavirus detection primers include: HPV6 detection primers, the forward primer sequence is shown in seq ID No. 175, and the rear primer sequence is shown in seq ID No. 176; HPV11 detection primers, the forward primer sequence is shown in seq ID No. 177, and the rear primer sequence is shown in seq ID No. 178; HPV40 detection primers, the forward primer sequence is shown in seq ID No. 179, and the rear primer sequence is shown in seq ID No. 180; HPV42 detection primers, the forward primer sequence is shown in seq ID No. 181, and the rear primer sequence is shown in seq ID No. 182; HPV43 detection primers, the forward primer sequence is shown in seq ID No. 183, and the rear primer sequence is shown in seq ID No. 184; HPV54 detection primers, the forward primer sequence is shown in seq ID No. 185, and the rear primer sequence is shown in seq ID No. 186; HPV61 detection primers, the forward primer sequence is shown in seq ID No. 187, and the rear primer sequence is shown in seq ID No. 188; HPV72 detection primers, the forward primer sequence is shown in seq ID No. 189, and the rear primer sequence is shown in seq ID No. 190; HPV81 detection primers, the forward primer sequence is shown in seq ID No. 191, and the rear primer sequence is shown in seq ID No. 192; HPV89 detection primers, the forward primer sequence is shown in seq ID No. 193, and the rear primer sequence is shown in seq ID No. 194; Other DNA virus detection primers include: Human herpesvirus type 1 detection primers, the forward primer sequence is shown in seq ID No. 209, and the rear primer sequence is shown in seq ID No. 210; Human herpesvirus type II detection primers, the forward primer sequence is shown in seq ID No. 211, and the rear primer sequence is shown in seq ID No. 212; Epstein-Barr virus detection primers, the forward primer sequence is shown in seq ID No. 213, and the rear primer sequence is shown in seq ID No. 214; Cytomegalovirus detection primers, the forward primer sequence is shown in seq ID No. 215, and the rear primer sequence is shown in seq ID No. 216; Molluscum contagiosum virus detection primers, the forward primer sequence is shown in seq ID No. 217, and the rear primer sequence is shown in seq ID No. 218; Hepatitis B virus detection primers, the forward primer sequence is shown in seq ID No. 219, and the rear primer sequence is shown in seq ID No. 220; Human polyomavirus type 1 detection primers, the forward primer sequence is shown in seq ID No. 221, and the rear primer sequence is shown in seq ID No. 222; Human polyomavirus type 2 detection primers, the forward primer sequence is shown in seq ID No. 223, and the rear primer sequence is shown in seq ID No. 224; Varicella-zoster virus detection primers, the forward primer sequence is shown in seq ID No. 225, and the rear primer sequence is shown in seq ID No. 226; RNA virus detection primers include: Human immunodeficiency virus type I detection primers, the forward primer sequence is shown in seq ID No. 125, and the rear primer sequence is shown in seq ID No. 126; Human immunodeficiency virus type II detection primers, the forward primer sequence is shown in seq ID No. 127, and the rear primer sequence is shown in seq ID No. 128; Hepatitis A virus detection primers, the forward primer sequence is shown in seq ID No. 129, and the rear primer sequence is shown in seq ID No. 130; Hepatitis C virus detection primers, the forward primer sequence is shown in seq ID No. 131, and the rear primer sequence is shown in seq ID No. 132; Human T-lymphotropic virus type 1 detection primers, the forward primer sequence is shown in seq ID No. 133, and the rear primer sequence is shown in seq ID No. 134; The fungus detection primers include: Candida albicans detection primers, the forward primer sequence is shown in seq ID No. 105, and the rear primer sequence is shown in seq ID No. 106; Primers for detecting Candida tropicalis, the forward primer sequence is shown in seq ID No. 107, and the rear primer sequence is shown in seq ID No. 108; Candida glabrata detection primers, the forward primer sequence is shown in seq ID No. 109, and the rear primer sequence is shown in seq ID No. 110; Candida krusei detection primers, the forward primer sequence is shown in seq ID No. 111, and the rear primer sequence is shown in seq ID No. 112; Candida dunovica detection primers, the forward primer sequence is shown in seq ID No. 117, and the rear primer sequence is shown in seq ID No. 118; The parasite detection primers include: Trichomonas vaginalis detection primers, the forward primer sequence is shown in seq ID No. 119, and the rear primer sequence is shown in seq ID No. 120; Entamoeba histolytica detection primers: the forward primer sequence is shown in seq ID No. 121, and the rear primer sequence is shown in seq ID No. 122; Toxoplasma gondii detection primers: the forward primer sequence is shown in seq ID No. 123, and the rear primer sequence is shown in seq ID No.

124.

2. The primer set for high-throughput sequencing of pathogens targeting reproductive tract infections according to claim 1, characterized in that: Also included are the following detection primers: HPV6 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 291, and the rear primer sequence is shown in seq ID No. 292; HPV11 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 293, and the rear primer sequence is shown in seq ID No. 294; HPV40 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 295, and the rear primer sequence is shown in seq ID No. 296; HPV42 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 297, and the rear primer sequence is shown in seq ID No. 298; HPV43 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 299, and the rear primer sequence is shown in seq ID No. 300; HPV54 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 301, and the rear primer sequence is shown in seq ID No. 302; HPV61 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 303, and the rear primer sequence is shown in seq ID No. 304; HPV70 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 305, and the rear primer sequence is shown in seq ID No. 306; HPV72 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 307, and the rear primer sequence is shown in seq ID No. 308; HPV81 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 309, and the rear primer sequence is shown in seq ID No. 310; HPV89 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 311, and the rear primer sequence is shown in seq ID No. 312; HPV34 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 313, and the rear primer sequence is shown in seq ID No. 314; HPV44 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 315, and the rear primer sequence is shown in seq ID No. 316; HPV57 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 317, and the rear primer sequence is shown in seq ID No. 318; HPV69 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 319, and the rear primer sequence is shown in seq ID No. 320; HPV71 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 321, and the rear primer sequence is shown in seq ID No. 322; HPV83 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 323, and the rear primer sequence is shown in seq ID No. 324; HPV84 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 325, and the rear primer sequence is shown in seq ID No.

326.

3. The primer set for high-throughput sequencing targeting reproductive tract infection pathogens according to claim 1, characterized in that: Also includes: The detection primers for the drug resistance gene parC, the forward primer sequence is shown in seq ID No. 229, and the rear primer sequence is shown in seq ID No. 230; The detection primers for the drug resistance gene penA, the forward primer sequence is shown in seq ID No. 233, and the rear primer sequence is shown in seq ID No. 234; The detection primers for the drug resistance gene ftsX, the forward primer sequence is shown in seq ID No. 235, and the rear primer sequence is shown in seq ID No. 236; The detection primers for the drug resistance gene PBP2x, the forward primer sequence is shown in seq ID No. 237, and the rear primer sequence is shown in seq ID No. 238; The detection primers for the drug resistance gene gyrA, the forward primer sequence is shown in seq ID No. 239, and the rear primer sequence is shown in seq ID No. 240; The detection primers for the drug resistance gene grlA, the forward primer sequence is shown in seq ID No. 241, and the rear primer sequence is shown in seq ID No. 242; The detection primers for the drug resistance gene graS, the forward primer sequence is shown in seq ID No. 243, and the rear primer sequence is shown in seq ID No. 244; The detection primers for the drug resistance gene rpoB, the forward primer sequence is shown in seq ID No. 245, and the rear primer sequence is shown in seq ID No. 246; The detection primers for the drug resistance gene ntr6, the forward primer sequence is shown in seq ID No. 247, and the rear primer sequence is shown in seq ID No. 248; The detection primers for the drug-resistant gene ERG11, the forward primer sequence is shown in seq ID No. 249, and the rear primer sequence is shown in seq ID No.

250.

4. The primer set for high-throughput sequencing targeting reproductive tract infection pathogens according to claim 1, characterized in that: Also includes at least one of the following pathogen detection primers: Prevotella detection primers, the forward primer sequence is shown in seq ID No. 15, and the rear primer sequence is shown in seq ID No. 16; Enterobacter hallii detection primers, the forward primer sequence is shown in seq ID No. 17, and the rear primer sequence is shown in seq ID No. 18; Pseudomonas detection primers, the forward primer sequence is shown in seq ID No. 77, and the rear primer sequence is shown in seq ID No. 78; HPV detection primers: HPV34 detection primers, the forward primer sequence is shown in seq ID No. 195, and the rear primer sequence is shown in seq ID No. 196; HPV44 detection primers, the forward primer sequence is shown in seq ID No. 197, and the rear primer sequence is shown in seq ID No. 198; HPV57 detection primers, the forward primer sequence is shown in seq ID No. 199, and the rear primer sequence is shown in seq ID No. 200; HPV69 detection primers, the forward primer sequence is shown in seq ID No. 201, and the rear primer sequence is shown in seq ID No. 202; HPV71 detection primers, the forward primer sequence is shown in seq ID No. 203, and the rear primer sequence is shown in seq ID No. 204; HPV83 detection primers, the forward primer sequence is shown in seq ID No. 205, and the rear primer sequence is shown in seq ID No. 206; HPV84 detection primers, the forward primer sequence is shown in seq ID No. 207, and the rear primer sequence is shown in seq ID No.

208.

5. The primer set for high-throughput sequencing targeting pathogens of reproductive tract infections according to claim 1, characterized in that: It also includes an internal standard sequence, the nucleic acid sequence of the internal standard sequence is shown as seq ID No.251 as the front primer sequence, and the rear primer sequence is shown as seq ID No.252 as the back primer sequence.

6. The primer set for high-throughput sequencing targeting reproductive tract infection pathogens according to claim 1, characterized in that: The detection primers are all connected to a linker sequence; the nucleic acid sequence of the linker sequence is shown in seq ID No.327 for the front primer and in seq ID No.328 for the rear primer.

7. A kit, characterized in that A primer set for high-throughput sequencing targeting reproductive tract infection pathogens comprising any one of claims 1 to 6.

8. The kit according to claim 7, characterized in that It also includes gDNA digestion reaction reagents, one-strand synthesis reaction reagents, first-step amplification reaction reagents, and second-step amplification reaction reagents; The reagent components of the gDNA digestion reaction include: gDNA wiper Mix reagent; The reagent components of the single-strand synthesis reaction include: RT Mix reagent, Enzyme Mix reagent, nuclease-free water and Random Primers reagent; The reagent components of the first step amplification reaction include: reaction buffer, nuclease-free water, multiplex amplification enzyme and PIC reagent; The reagent components of the second step amplification reaction include: Hifi Mix reagent.

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